During the current funding period, we have continued our studies of EBP67, a 67 kDa elastin-binding protein found on elastin-producing cells. Unlike signaling or adhesion receptors, EBP67 participates in the assembly of the elastic fiber by mediating the transfer of tropoelastin to assembly sites on microfibrils. Based on many common features with intracellular chaperones, we propose that EBP67 functions as a molecular chaperone for tropoelastin. Like many chaperones, EBP67 preferentially binds to hydrophobic sequences, has evolved a mechanism for selected release of protein ligand, and participates in oligomeric assembly. EBP67 also exhibits many properties that suggest it might play an imperant role in the intracellular management of tropoelastin secretion, such as intracellular colocalization with tropoelastin and interactions with cytoskeletal components that act as motors for moving EBP67-tropoelastin complexes. We have also found that a 61 kDa protein that purifies with EBP67 is TCP-1, itself a major component of cytoplasmic chaperones. The possibility that EBP67 may be a chaperone for a secreted protein is an important new extension of the chaperone paradigm. Another important observation made during the current funding period was that the integrin alpha-v-beta3 binds to the microfibrillar protein fibrillin. This raises the interesting possibility that alpha-v-beta-3 is an integral component of elastic fiber assembly sites on the plasma membrane. These assembly sites morphologically resemble focal contacts as characterized by dense areas of membrane with an accumulation of cytoskeletal elements on the cytoplasmic side. Given the propensity of alpha-v-beta3 to associate into focal contacts and to interact with the cytoskeleton, we hypothesize that this integrin plays an important role in defining assembly sites through its bridging interactions between the intracellular cytoskeleton and fibrillin on the plasma membrane. Our future objectives are to continue to characterize EBP67 and its accessory proteins and to explore EBP67's possible function as a molecular chaperone. We will also examine a possible role for alpha-v-beta3 integrin in elastic fiber assembly. Finally, we wish to better define the elastic fiber assembly site on the cell surface. Our specific aims are: l) To further characterize EBP67 and its accessory proteins; 2) To characterize the structural motifs recognized by EBP67 on protein ligands; 3) To investigate the intracellular location of EBP67 in the secretory pathway and characterize its role in the secretion of tropoelastin; and 4) Determine whether the alpha-v-beta3 integrin plays a role in elastic fiber assembly through its interactions with fibrillin.